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Scientific Report 2008


Molecular Biology




Structural Biology of Viral Proteins, Molecular Assemblies, and the Immune System


I.A. Wilson, R.L. Stanfield, J. Stevens, X. Zhu, M.A. Adams, C.H. Bell, R.M.F. Cardoso, J. Carlson, P.J. Carney, S. Connelly, A.L. Corper, T. Cross, X. Dai, E.W. Debler, W.L. Densley, B.J. Droese, D.C. Ekiert, M.-A. Elsliger, S. Ferguson, Z. Fulton, B.W. Han, G.W. Han, M. Hong, M.J. Jimenez-Dalmaroni, R.N. Kirchdoerfer, J.R. Mikolosko, R. Pejchal, G.P. Porter,
A. Schiefner, D.A. Shore, R.S. Stefanko, J.A. Vanhnasy,
P. Verdino, R. Xu, X. Xu, S.I. Yoon

To understand the immune response to invading pathogens, such as bacteria and viruses, we focus on the structure-function relationships of immune molecules and their microbial targets. These structural results are especially useful in the design of drugs and vaccines that target the pathogens and protect the host.

The Innate Immune System

To enhance our understanding of the molecular biology of innate immune receptors, we are investigating the activation requirements of γδ T cells. In collaboration with W. Havran, Department of Immunology and Microbial Science, we determined the structure of junctional adhesion molecule-like (JAML), the γδ T cell—specific costimulatory molecule, in complex with coxsackievirus-adenovirus receptor, its endogenous ligand (Fig. 1). The structure revealed an unusually hydrophilic complex interface that suggests potential mechanisms for receptor triggering. Upon JAML engagement, different kinases are then recruited at the JAML intracellular domain to activate kinase signaling cascades, production of cytokines and chemokines, and, ultimately, proliferation of γδ T cells.


Fig. 1. Crystal structure of JAML in complex with coxsackievirus-adenovirus receptor (CAR). JAML and the receptor interact with their membrane-distal D1 immunoglobulin domains in a face-to-face β-sheet interaction.

Toll-like receptors are cell-surface receptors that detect invading microbes by recognizing a variety of pathogen-associated molecular patterns, including bacterial cell walls and viral nucleic acids. To reveal structural mechanisms involved in activation and regulation of these receptors, we have expressed the extracellular domain of human Toll-like receptor 4 with myeloid differentiation protein-2 for structural studies. This immune complex binds bacterial lipopolysaccharides, ultimately leading to sepsis.

Among the pattern-recognition molecules, the intracellular Nod-like receptors also act as key mediators of innate immunity and of the inflammatory response to microbial infections. Mutations in the genes for these receptors are associated with chronic inflammatory barrier diseases, such as Crohn's disease and bronchial asthma. We have expressed and purified Nod1 and Nod2 proteins for crystallization. The studies on the Toll- and Nod-like receptors are collaborations with R. Ulevitch, Department of Immunology and Microbial Science, and B. Beutler, Department of Genetics.

Nonmammalian Innate and Adaptive Immunity

Variable lymphocyte receptors (VLRs) play a key role in recognition of antigens in the adaptive immune response of jawless vertebrates. In collaboration with M.D. Cooper, Emory University School of Medicine, Atlanta, Georgia, we determined the crystal structure of the lamprey VLR2913 ectodomain to 2.1-Å resolution. The VLR folds into a horseshoe-shaped, solenoidal assembly of 5 leucine-rich repeats. Although the antigen for VLR2913 is unknown, the highly similar VLR4 interacts with the anthrax antigen, bacillus collagen-like protein of anthracis (BclA). We have modeled VLR4 with Modeller, on the basis of the VLR2913 structure, and then used Autodock 4.0 software to dock the VLR4 model to BclA, in collaboration with A.J. Olson and G.M. Morris, Department of Molecular Biology (Fig. 2). The docking results suggest that the concave surface of VLR4 is the recognition site for BclA.


Fig. 2. Model of the complex formed by lamprey VLR4 and BclA. The VLR4 was modeled by using the computer program Modeller, with the lamprey VLR2913 crystal structure as a template. The VLR4 model was then tested for the lowest energy docking orientation with BclA by using Autodock software.

Classical and Nonclassical MHC and T-Cell Receptor Signaling

T-cell receptors (TCRs) recognize peptide antigen displayed on the surface of antigen-presenting cells by MHC molecules. Coreceptor molecules, such as CD8αβ and CD4, provide costimulatory signals that are required for full T-cell activation. To ascertain the structural basis for the coreceptor function of CD8αβ and understand the mechanisms that underlie T-cell activation, we used a single-chain MHC-CD8αβ construct to determine the structure of the CD8αβ-MHC-peptide complex (Fig. 3).


Fig. 3. Crystals of the MHC class I—peptide—CD8αβ single-chain complex.

To explore potential mechanisms of diabetogenesis, we are investigating whether TCRs recognize the oxyanion hole at P9 that is present only in the diabetes-associated MHC I-Ag7. We isolated a TCR hybridoma (21.30) that is sensitive to the presence or absence of a negatively charged P9 peptide residue and determined the crystal structure to 3.5-Å resolution of TCR 21.30— I-Ag7—HEL9-27, where P9 is a glycine. Surprisingly, the structure revealed that TCR 21.30 does not directly contact the I-Ag7 P9 pocket. Experiments are under way to ascertain the TCR sensitivity to this residue position.

MHC molecules play a critical role in initiating cell-mediated immunity by presenting both foreign antigens and self-antigens. Efficient loading of peptide antigens on MHC class I molecules requires proteins in the endoplasmic reticulum collectively known as the peptide-loading complex. The complex consists of the transporter associated with antigen processing, tapasin, calreticulin, calnexin, ERp57, and an MHC class I molecule. Currently, we are focusing on the structural and biochemical characterization of the transporter, tapasin, and the peptide-free form of MHC class I molecules. Recombinant expression of tapasin from several species, as well as recombinant expression of the transporter, have provided valuable tools for analyzing the structure, function, and assembly of the peptide-loading complex. Biochemical and biophysical techniques are being used to provide structural models for MHC class Ia folding and antigen presentation. The MHC and TCR studies are a collaboration with L. Teyton, Department of Immunology and Microbial Science.

CD1 molecules are MHC class I antigen-presenting molecules that present lipids, glycolipids, and lipopeptides to effector T cells. CD1 molecules are involved in host defense and also have immunoregulatory functions. Glycolipids presented by CD1d stimulate natural killer T cells, which are of clinical interest because they rapidly secrete cytokines that either promote or suppress different immune responses. On the basis of our structural studies, C.-H. Wong and his group, Department of Chemistry, synthesized a series of glycolipids that are more potent than other glycolipids tested previously and have increased efficacy in T-cell assays. Structures for 2 of the most stimulating glycolipids in complex with CD1d have revealed that loading and anchoring of the lipids are the key determinants for effective lipid presentation and subsequent T-cell stimulation.

Influenza Virus

To aid in design of vaccines and drugs to prevent future influenza pandemics, we are studying proteins from different influenza strains as part of a consortium funded by the National Institute of Allergy and Infectious Diseases. The 1918 flu pandemic was the most devastating epidemic in recorded world history, and efforts are ongoing to target the neuraminidase of the 1918 influenza virus in structure-based drug design. We have determined crystal structures for the 1918 N1 neuraminidase from crystals with an unusual defect called a "lattice translocation.” Although the successful use of twinned data for structure determination has become relatively routine in recent years, structure determination with lattice-translocation defects has only been previously reported for 5 structures. In addition, structures of the 1918 neuraminidase in complex with zanamivir (Relenza) and oseltamivir (Tamiflu) have revealed new cavities for drug binding (Fig. 4) and how the presence or absence of different ions can affect the overall assembly of the neuraminidase tetramer.


Fig. 4. Molecular surface of the 1918 influenza virus N1 neuraminidase active site. Zanamivir (ball-and-stick model) has been docked into the unliganded neuraminidase structure on the basis of the drug's position in the zanamivir-neuraminidase complex. This superposition reveals a large, unoccupied cavity close to the zanamivir binding site that may be an excellent target for the design of inhibitors.

The H5N1 avian influenza viruses currently circulating in Asia, Europe, and Africa are extremely virulent in humans, causing severe disease and often death. H5N1 viruses are not readily transmitted among humans, possibly because of differences in the receptor specificity of the hemagglutinin viral fusion protein. To investigate structural changes critical for receptor switching from avian to human specificity, we have developed a baculovirus display platform that will enable us to test large libraries of hemagglutinin mutants for binding to immobilized glycans or human bronchial cell monolayers. The receptor specificity of the selected variants will be determined by using glycan arrays, and structural changes associated with receptor switching will be characterized by using x-ray crystallography. In collaboration with G.J. Tobin, Biological Mimetics, Inc., Frederick, Maryland, the full-length hemagglutinin from an outbreak of influenza in Wyoming and HA2 hemagglutinin fragments from outbreaks in South Carolina in 1918 (H1H1) and Vietnam in 2003 (H5N1) are being expressed for structural studies of conformations before and after fusion. In addition, many collaborations are ongoing to investigate the neutralization of H1N1 and H5N1 viruses by monoclonal antibodies.

HIV Type 1 Vaccine

The need for an effective HIV vaccine is greater than ever as the virus continues to devastate areas of the world such as sub-Saharan Africa. As a part of our vaccine development efforts, we are studying the viral envelope "spikes” composed of heterotrimeric complexes of gp120 and gp41. Upon binding receptors CD4 and CXCR4/CCR5, the trimer undergoes as yet uncharacterized conformational changes that lead to fusion of the viral membrane with the target cell, initiating infection. Crystallization of the trimer in the prefusion state will enable a detailed understanding of its antibody epitope landscape and reveal how neutralizing antibodies can recognize this evolutionarily moving target.

We recently determined the crystal structure of a human monoclonal antibody, F425-B4e8 (B4e8), that cross-reacts with the gp120 V3 region of primary viral isolates from subtypes B, C, and D. The B4e8 Fab in complex with the 24mer V3 peptide RP142 showed that the antibody recognizes a novel V3 loop conformation with a 5-residue α-turn around the conserved GPGRA apex of the β-hairpin loop (Fig. 5). The Fab interacts primarily through main-chain interactions with major contacts to only 2 V3 peptide side chains, explaining how B4e8 can accommodate sequence variation within V3 and hence can neutralize different isolates of HIV type 1.


Fig. 5. Structure of F425-B4e8 Fab in complex with a peptide (red) representing the V3 region of HIV type 1 gp120. B4e8 is unusually broad in its neutralization of different HIV type 1 viral isolates, and the peptide conformation recognized has an unusual α-turn around the tip of the loop.

Our research on HIV is done in collaboration with D.R. Burton, Department of Immunology and Microbial Science; P.E. Dawson, Department of Cell Biology; C.-H. Wong, Department of Chemistry; J.K. Scott, Simon Fraser University, Burnaby, British Columbia; J. Moore, Weill Medical College of Cornell University, New York, New York; H. Katinger, R. Kunert, and G. Stiegler, University für Bodenkultur, Vienna, Austria; R. Wyatt and P. Kwong, Vaccine Research Center, National Institutes of Health, Bethesda, Maryland; W. Olson, and K. Kang, Progenics Pharmaceuticals, Inc., Tarrytown, New York; the National Institutes of Health, Bethesda, Maryland; and the Neutralizing Antibody Consortium of the International AIDS Vaccine Initiative, New York, New York.

Blue Fluorescent Antibodies

EP2-19G2, an antibody to a trans-stilbene, has bright blue luminescence and has been used as a biosensor in various applications. By extensive biophysical characterization of the stilbene-antibody complex, we found that the prolonged luminescence is due to a charge-transfer excited complex of an anionic stilbene and a cationic, parallel π-stacked tryptophan. Upon charge recombination, this complex generates exceptionally bright blue light. Formation of the complex is supported by a deep ligand-binding pocket, which in turn is due to a noncanonical interface between the 2 variable antibody subunits. These studies are collaborations with R.A. Lerner, K.D. Janda, and P.G. Schultz, Department of Chemistry; D.P. Millar, Department of Molecular Biology; and H.B. Gray, California Institute of Technology, Pasadena, California.

Histone Deacetylases

Histone deacetylases catalyze removal of the acetyl group from amino-terminal lysine residues in histones, resulting in chromatin condensation and transcriptional repression. Inhibitors of histone deacetylases are a widely used treatment for many types of cancer. In recent years, these compounds have been emerging as a potential therapy for neurodegenerative disorders, such as Friedreich ataxia, an inherited disease that affects the nervous system and results in muscle weakness, heart disease, and speech difficulties. In collaboration with J.M. Gottesfeld, Department of Molecular Biology, and with support from the Friedreich's Ataxia Research Alliance, Springfield, Virginia, we are expressing several histone deacetylases for determinations of crystal structures of the enzymes in complex with inhibitors.

Joint Center for Structural Genomics

The Joint Center for Structural Genomics is a large consortium of scientists from Scripps Research; the Stanford Synchrotron Radiation Laboratory; the University of California, San Diego; the Burnham Institute for Medical Research; and the Genomics Institute of the Novartis Research Foundation. The center is funded by the Protein Structure Initiative of the National Institute of General Medical Sciences. Its purpose is high-throughput structure determination of large families of proteins with no or limited structural representatives, biologically important targets that are conserved as the central machinery of life, the complete proteome from Thermotoga maritima, metagenomic and human microbiome targets, and other targets suggested by the community. To date, the members of the consortium have pioneered many novel high-throughput methods and technologies applicable to structural biology and have determined more than 665 unique structures, including more than 200 novel structures in the past year.

Publications

Astronomo, R.D., Lee, H.K., Scanlan, C.N., Pantophlet, R., Huang, C.Y., Wilson, I.A., Blixt, O., Dwek, R.A., Wong, C.-H., Burton, D.R. A glycoconjugate antigen based on the recognition motif of a broadly neutralizing human immunodeficiency virus antibody, 2G12, is immunogenic but elicits antibodies unable to bind to the self glycans of gp120. J. Virol. 82:6359, 2008.

Bell, C.H., Pantophlet, R., Schiefner, A., Cavacini, L.A., Stanfield, R.L., Burton, D.R., Wilson, I.A. Structure of antibody F425-B4e8 in complex with a V3 peptide reveals a new binding mode for HIV-1 neutralization. J. Mol. Biol. 375:969, 2008.

Burley, S.K., Joachimiak, A., Montelione, G.T., Wilson, I.A. Contributions to the NIH-NIGMS Protein Structure Initiative from the PSI Production Centers. Structure 16:5, 2008.

Burton, D.R., Wilson, I.A. Immunology: square-dancing antibodies. Science 317:1507, 2007.

Debler, E.W., Müller, R., Hilvert, D., Wilson, I.A. Conformational isomerism can limit antibody catalysis. J. Biol. Chem. 283:16554, 2008.

Debler, E.W., Kaufmann, G.F., Meijler, M.M., Heine, A., Mee, J.M., Pljevaljcic, G., Di Bilio, A.J., Schultz, P.G., Millar, D.P., Janda, K.D., Wilson, I.A., Gray, H.B., Lerner, R.A. Deeply inverted electron-hole recombination in a luminescent antibody-stilbene complex. Science 319:1232, 2008.

Huang, C.C., Lam, S.N., Acharya, P., Tang, M., Xiang, S.H., Hussan, S.S., Stanfield, R.L., Robinson, J., Sodroski, J., Wilson, I.A., Wyatt, R., Bewley, C.A., Kwong, P.D. Structures of the CCR5 N terminus and of a tyrosine-sulfated antibody with HIV-1 gp120 and CD4. Science 317:1930, 2007.

Huang, S., Romanchuk, G., Pattridge, K., Lesley, S.A., Wilson, I.A., Matthews, R.G., Ludwig, M. Reactivation of methionine synthase from Thermotoga maritima (TM0268) requires the downstream gene product TM0269. Protein Sci. 16:1588, 2007.

Johnson, S.M., Connelly, S., Wilson, I.A., Kelly, J.W. Biochemical and structural evaluation of highly selective 2-arylbenzoxazole-based transthyretin amyloidogenesis inhibitors. J. Med. Chem. 51:260, 2008.

Kozbial, P., Xu, Q., Chiu, H.J., et al. Crystal structures of MW1337R and lin2004: representatives of a novel protein family that adopt a four-helical bundle fold. Proteins 71:1589, 2008.

Krishna, S.S., Tautz, L., Xu, Q., et al. Crystal structure of NMA1982 from Neisseria meningitidis at 1.5 Å resolution provides a structural scaffold for nonclassical, eukaryotic-like phosphatases. Proteins 69:415, 2007.

Mathews, I.I., McMullan, D., Miller, M.D., et al. Crystal structure of 2-keto-3-deoxygluconate kinase (TM0067) from Thermotoga maritima at 2.05 Å resolution. Proteins 70:603, 2008.

Menendez, A., Calarese, D.A., Stanfield, R.L., Chow, K.C., Scanlan, C.N., Kunert, R., Katinger, H., Burton, D.R., Wilson, I.A., Scott, J.K. A peptide inhibitor of HIV-1 neutralizing antibody 2G12 is not a structural mimic of the natural carbohydrate epitope on gp120. FASEB J. 22:1380, 2008.

Premkumar, L., Rife, C.L., Sri Krishna, S., et al. Crystal structure of TM1030 from Thermotoga maritima at 2.3 Å resolution reveals molecular details of its transcription repressor function. Proteins 68:418, 2007.

Sanguineti, S., Centeno Crowley, J.M., Lodeiro Merlo, M.F., Cerutti, M.L., Wilson, I.A., Goldbaum, F.A., Stanfield, R.L., de Prat-Gay, G. Specific recognition of a DNA immunogen by its elicited antibody. J. Mol. Biol. 370:183, 2007.

Saphire, E.O., Montero, M., Menendez, A., van Houten, N.E., Irving, M.B., Pantophlet, R., Zwick, M.B., Parren, P.W., Burton, D.R., Scott, J.K., Wilson, I.A. Structure of a high-affinity "mimotope” peptide bound to HIV-1-neutralizing antibody b12 explains its inability to elicit gp120 cross-reactive antibodies. J. Mol. Biol. 369:696, 2007.

Slabinski, L., Jaroszewski, L., Rodrigues, A.P., Rychlewski, L., Wilson, I.A., Lesley, S.A., Godzik, A. The challenge of protein structure determination—lessons from structural genomics. Protein Sci. 16:2472, 2007.

Slabinski, L., Jaroszewski, L., Rychlewski, L., Wilson, I.A., Lesley, S.A., Godzik, A. XtalPred: a web server for prediction of protein crystallizability. Bioinformatics 23:3403, 2007.Stoll, R., Lee, B.M., Debler, E.W., Laity, J.H., Wilson, I.A., Dyson, H.J., Wright, P.E. Structure of the Wilms tumor suppressor protein zinc finger domain bound to DNA. J. Mol. Biol. 372:1227, 2007.

Structural Genomics Consortium; China Structural Genomics Consortium; Northeast Structural Genomics Consortium; Gräslund, S., Nordlund, P., Weigelt, J., et al. Protein production and purification. Nat. Methods 5:135, 2008.

Wei, C.J., Xu, L., Kong, W.P., Shi, W., Canis, K., Stevens, J., Yang, Z.Y., Dell, A., Haslam, S.M., Wilson, I.A., Nabel, G.J. Comparative efficacy of neutralizing antibodies elicited by recombinant hemagglutinin proteins from avian H5N1 influenza virus. J. Virol. 82:6200, 2008.

Xu, Q., Kozbial, P., McMullan, D., et al. Crystal structure of an ADP-ribosylated protein with a cytidine deaminase-like fold, but unknown function (TM1506), from Thermotoga maritima at 2.70 Å resolution. Proteins 71:1546, 2008.

Xu, Q., Saikatendu, K.S., Krishna, S.S., et al. Crystal structure of MtnX phosphatase from Bacillus subtilis at 2.0 Å resolution provides a structural basis for bipartite phosphomonoester hydrolysis of 2-hydroxy-3-keto-5-methylthiopentenyl-1-phosphate. Proteins 69:433, 2007.

Zajonc, D.M., Savage, P.B., Bendelac, A., Wilson, I.A., Teyton, L. Crystal structures of mouse CD1d-iGb3 complex and its cognate Vα 14 T cell receptor suggest a model for dual recognition of foreign and self glycolipids. J. Mol. Biol. 377:1104, 2008.

Zajonc, D.M., Wilson, I.A. Architecture of CD1 proteins. Curr. Top. Microbiol. Immunol. 314:27, 2007.

Zubieta, C., Joseph, R., Krishna, S.S., et al. Identification and structural characterization of heme binding in a novel dye-decolorizing peroxidase, TyrA. Proteins 69:234, 2007.

Zubieta, C., Krishna, S.S., Kapoor, M., et al. Crystal structures of two novel dye-decolorizing peroxidases reveal a β -barrel fold with a conserved heme-binding motif. Proteins 69:223, 2007.

Zubieta, C., Krishna, S.S., McMullan, D., et al. Crystal structure of homoserine O-succinyltransferase from Bacillus cereus at 2.4 Å resolution. Proteins 68:999, 2007.



 

Ian A. Wilson, D.Phil.
Professor

Robyn L. Stanfield, Ph.D.
Associate Professor



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